Identification of a Virulent Newcastle Disease Virus Strain Isolated from Pigeons (Columbia livia) in Northeastern Brazil Using Next-Generation Genome Sequencing

Abstract

Newcastle disease virus (NDV), also known as avian paramyxoviruses 1 (APMV-1) is among the most important viruses infecting avian species. Given its widespread circulation, there is a high risk for the reintroduction of virulent strains into the domestic poultry industry, making the surveillance of wild and domestic birds a crucial process to appropriately respond to novel outbreaks. In the present study, we investigated an outbreak characterized by the identification of sick pigeons in a large municipality in Northeastern Brazil in 2018. The affected pigeons presented neurological signs, including motor incoordination, torticollis, and lethargy. Moribund birds were collected, and through a detailed histopathological analysis we identified severe lymphoplasmacytic meningoencephalitis with perivascular cuffs and gliosis in the central nervous system, and lymphoplasmacytic inflammation in the liver, kidney, and intestine. A total of five pigeons tested positive for NDV, as assessed by rRT-PCR targeted to the M gene. Laboratory virus isolation on Vero E6 cells confirmed infection, after the recovery of infectious NVD from brain and kidney tissues. We next characterized the isolated NDV/pigeon/PE-Brazil/MP003/2018 by next-generation sequencing (NGS). Phylogenetic analysis grouped the virus with other NDV class II isolates from subgenotype VI.2.1.2, including two previous NDV isolates from Brazil in 2014 and 2019. The diversity of aminoacid residues at the fusion F protein cleavage site was analyzed identifying the motif RRQKR↓F, typical of virulent strains. Our results all highlight the importance of virus surveillance in wild and domestic birds, especially given the risk of zoonotic NDV.

Keywords: Newcastle disease virus; avian paramyxovirus 1; domestic birds; pigeons.

Figure 1

Severe lymphoplasmacytic inflammatory infiltrate expanding the perivascular space and severe vasculitis in leptomeningeal vessels (arrows). Note moderate diffuse vacuolization in the neuropil of the occipital cortex. HE staining, objective magnification 10× (A). Blood vessels from the leptomeninges presenting perivascular cuffs are composed mainly of lymphocytes, plasma cells, and rare macrophages (arrows). HE staining, objective magnification 40× (B). Mild infiltration of lymphocytes inflammatory cells in the meningeal space and superficial cortex (arrows), mainly composed of lymphocytes, plasma cells, and a few macrophages, diffuse severe gliosis in the inner gray matter. HE staining, objective magnification 10× (C). Higher magnification of C where diffuse gliosis and neuronal necrosis are characterized by shrunken, eosinophilic, and pyknotic neurons (arrows). HE staining, objective magnification 40× (D).

Figure 2

Phylogenetic characterization of NDV/pigeon/PE-Brazil/MP003/2018. Phylogenetic analysis was based on the full-length nucleotide sequence of NDV. The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model. The tree with the highest log likelihood (−18,158.31) is shown. Our analysis classified the NDV/pigeon/PE-Brazil/MP003/2018 as genotype VI.2.1.2 strain (highlighted with a red square). The Roman numerals presented in the taxa names in the phylogenetic tree represent the respective sub/genotype for each isolate, followed by the GenBank Accession number, hostname, country of isolation, and year of isolation (if available).

Figure 3

Sequence analysis of F protein cleavage site in NDV isolates. The diversity of residues at each position of the fusion cleavage site was analyzed using WebLogo 3.1 (http://weblogo.threeplusone.com/create.cgi (accessed on 28 April 2022)). Frequency of amino acid residues from the F protein fusion cleavage site motif of virulent NDV natural isolates, representative of 1073 isolates (A). Frequency of amino acid residues from the F protein fusion cleavage site motif of avirulent NDV natural isolates, representative of 499 isolates (B). The motif ¹¹²RRQKR↓F¹¹⁷ from NDV/pigeon/PE-Brazil/MP003/2018 lies with virulent strains and is the most common motif observed from 801 strains as reported by Wang et al., 2017 [23].